Heparin affinity chromatography has been used extensively for the purification of stimulators of endothelial cell (EC) proliferation such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF). This technique has now been used to identify an apparently novel heparin-binding inhibitor of EC proliferation that is secreted by monocytes and macrophages. The heparin-binding EC inhibitor (HB-ECI) inhibits basal EC proliferation and antagonizes the mitogenic activity of bFGF for EC. The inhibition is non-toxic, reversible and is characterized by a change in EC morphology from cobblestone to fibroblastic in nature. HB-ECI does not inhibit smooth muscle cell proliferation. Neutralizing antibody and other studies indicate that HB-ECI is distinct from other EC inhibitors such as transforming growth factor-beta (TGF-beta), tumor necrosis factor-alpha (TNF-alpha), cartilage-derived inhibitor, thrombospondin and platelet factor-4. Preliminary characterization indicates that HB-ECI is a highly stable, cationic polypeptide of about 18 kDa. bFGF, an EC agonist, needs to bind to cell surface heparan sulfate proteoglycan (HSPG) in order to be mitogenic. As a heparin-binding factor, HB-ECI might also bind to EC surface HSPG. Thus, HB-ECI might antagonize the mitogenic activity of bFGF by competing for cell surface HSPG binding sites on EC. Macrophages accumulate in tumors and wounds, and are thought to modulate neo-vascularization via specific macrophage products such as bFGF, TGF-beta, TGF-alpha, PDGF and TNF-alpha. Since HB-ECI is secreted by monocytes and macrophages, it could play an important role in regulating angiogenesis associated with inflammation, repair and tumor growth. Given its potential as a modulator of angiogenesis, the major goals of the research are to purify and characterize HB-ECI, analyze its biological properties and clone its gene. The specific aims of the proposal are: 1. To purify and characterize the macrophage-derived heparin-binding endothelial cell inhibitor (HB-ECI) and its receptor and to prepare anti- HB-ECI antibodies for immuno-detection and neutralization studies; 2. To analyze the biological properties of HB-ECI in vitro and in vivo; such as target cell specificity and effects on EC proliferation, migration, matrix production, protease levels, tube formation and angiogenesis; 3. To analyze HB-ECI interactions with heparin, cell surface HSPG and HSPG in extracellular matrix; 4. To clone the HB-ECI gene and analyze HB-ECI expression in cells and tissue.